QC and filter seurat object

Parameter settings

Import Seurat object

Add filter meta data to Seurat object

Filter cells by rank plot

Empty droplets were determined with CellRanger V3.0.2 Lun et al., 2019 EmptyDrop heuristic. RNAse activity of granulocytes might be wrongly identified as empty cells by CellRanger.

Typical Sample A steep drop-off is indicative of good separation between the cell-associated barcodes and the barcodes associated with empty GEMs. A ideal barcode rank plot has a distincitve shape, which is referred to as a "cliff and knee".

Heterogeneous Sample Heterogeneous populations of cells in a sample result in two "cliff and knee" distributions. However, there should still be clear separation between the bacodes.

Compromised Sample Round curve and lack of steep cliff may indicate low sample quality or loss of single-cell behavior. This can be due to a wetting failure, premature cell lysis, or low cell viability.

Compromised Sample Defined cliff and knee, but the total number of barcodes detected may be lower than expected. This can be caused by a sample clog or inaccurate cell count.

Filter by cell_ranger class

Filter by UMI and Feature count

Cell Cycle Raw Seurat Object

Box plots

Cell cycle QC Seurat object

Normalize

Variable feature selection

Remove cell cycle variable feature

Scale data

Regress nCount_RNA (total UMI per cell) and pMT_RNA (mitochondrial UMI percentage)

Dim reduction

SingleR annotation

SingleR identifies marker genes from the reference and uses them to compute assignment scores (based on the Spearman correlation across markers) for each cell in the test dataset against each label in the reference. The label with the highest score is the assigned to the test cell, possibly with further fine-tuning to resolve closely related labels.

first.labels: Labels before fine-tuning
labels: Labels after fine-tuning
pruning: labels after pruning

Annotate all cells

Dim and Feature plots

Bar plot cluster overlap

Cluster marker

FACS marker

CD45 - Ptprc for Immune cells Erythroblast marker CD71 Tfrc
CD71 is an integral membrane protein that mediates the uptake of transferrin iron complexes

Histocompatibility Class I

H2-K1 - Class I histocompatibility antigen, kappa-B alpha chain
H2-D1 - Class I histocompatibility antigen, D-B alpha chain
H2-L1 - not found

Histocompatibility Class II

H2-Aa - Class II histocompatibility antigen, A-B alpha chain
H2-Ab1 - Class II histocompatibility antigen, A beta chain

H2-Eb1 - Class II histocompatibility antigen, I-E beta chain
H2-Eb2 - Class II histocompatibility antigen, E beta chain

T-cell TCR-CD3 complex

Trac - T cell receptor alpha constant
Trbc1 - T cell receptor beta constant 1
Trbc2 - T cell receptor beta constant 2
Trdc - T cell receptor delta constant
Trgc1 - T cell receptor gamma constant 1
Trgc2 - T cell receptor gamma constant 2

Cd247 - T-cell surface glycoprotein Cd3 zeta chain (Cd3z)
Cd3g - T-cell surface glycoprotein Cd3 gamma chain
Cd3e - T-cell surface glycoprotein Cd3 epsilon chain
Cd3d - T-cell surface glycoprotein Cd3 delta chain

B-cell Immonogloblin complex

Naive B-cells produce the following Ig classes:
Ighm - Immunoglobulin heavy constant mu (naive B-cells)
Ighd - Immunoglobulin heavy constant delta (naive B-cells)

Through isotope switching the following Ig classes can be produced:
Ighg1 - Immunoglobulin heavy constant gamma (Mouse with Igh1b have Igg2c isotope instead of Igg2a)
Ighg2a - Immunoglobulin heavy constant gamma (NA)
Ighg2b - Immunoglobulin heavy constant gamma
Ighg2c - Immunoglobulin heavy constant gamma
Ighg3 - Immunoglobulin heavy constant gamma
Igha - Immunoglobulin heavy constant alpha
Ighe - Immunoglobulin heavy constant epsilon (NA)

Igkc - Immunoglobulin kappa constant (light chain)
Iglc - Immunoglobulin lambda constant (light chain)

Save output

Its only possible to store two out of three count matrix. Data can be recovered from counts but not vice versa. However, data can not be removed and is prioretized before counts. Therefore we fill counts in the data slot so that it gets exported.

Write Seurat components

Session info